Resveratrol Ameliorates Alcoholic Fatty Liver By Activating AMPK- Lipin1 Signaling

Alcoholic liver disease (ALD) is a chronic liver disease caused by long-term heavy drinking. Alcohol and its metabolic products not only damage the liver, but also stimulate the formation of lipid and cause lipid metabolism disorders. Alcoholic fatty liver (AFL) is initial stage of ALD, may develop advanced liver diseases such as alcoholic fibrosis, cirrhosis, and even hepatocellular carcinoma with the continuation of alcohol abuse.Resveratrol (RSV) is a polyphenol compounds, mainly comes from the grape (red wine), giant knotweed, peanut, mulberry and other plants. It has a wide range of pharmacological effects, including anti-cancer, anti-oxidation, anti-inflammatory, anti-angiogenic, hepatoprotective, etc.In the present study, we evaluated the effects of RSV treatment on AFL in mice fed with ethanol Lieber-DeCarli liquid diet and investigated the potential molecular mechanism of RSV on lipid accumulation in HepG2 cells induced by oleic acid and alcohol. The main contents are as follows:1. Protective effects of RSV on AFL in miceMice were established model of AFL by chronic ethanol feeding (5days ad libitum oral feeding with the control Lieber-DeCarli liquid diet, and then 10 days ad libitum oral feeding with the ethanol Lieber-DeCarli liquid diet) plus a single binge ethanol feeding. Mice were randomly divided into six groups:control group fed with control Lieber-DeCarli liquid diet; model group fed with ethanol Lieber-DeCarli liquid diet; RSV groups fed with ethanol Lieber-DeCarli liquid diet and given different RSV doses (10,30,100 mg/kg) by gavage; positive drug group fed with ethanol Lieber-DeCarli liquid diet and given reduced glutathione (GSH,200mg/kg) by intraperitoneal injection. At the end of the experimental period, the final body weight and liver weight of each mouse were recorded to calculate liver index. The serum levels of triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the hepatic levels of TG, superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. The liver specimens were stained with hematoxylin and eosin (HE) for histological examination. The expression of adipose differentiation-related protein (ADRP) was detected by immunohistochemistry (IHC) method. The results showed that compared with control group, liver index, the serum levels of ALT, AST and TG, and the hepatic levels of TG and MDA significantly increased in model group, alcohol administration led to a significant decrease in the activities of SOD. Treatment with RSV significantly suppressed these effects. However, we found that RSV had no effects on the serum level of TG. HE staining revealed that high degree of hepatic steatosis, excessive lipid droplets had accumulated in hepatocytes in model group. Treatment of RSV led to a decrease in the formation of lipid droplets compared with model group. IHC analysis showed that after RSV administration, ADRP expression decreased compared with model group. These results suggested that RSV had protective effects on AFL in mice.2. The effects of RSV on expression of hepatic AMPK and Lipinl in AFL miceAMPK is the key factor in the regulation of lipid metabolism, which plays an important role in the development of the AFL. Lipinl is a newly discovered regulation factor which has a dual role in lipid metabolism. On the one hand, lipinl is as a phosphatidate phosphatase (PAP) enzyme, which catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol required for lipid synthesis, on the other hand, it also acts as a transcriptional coactivator by interacting with peroxisome proliferator- activated receptory (PPARγ) coactivator-1α(PGC-1α) and PPARa to promote fatty acid oxidation. The expression of hepatic AMPK and lipinl in AFL mice was detected by western blot method. The results showed that the level of p-AMPK significantly decreased and the expression of lipinl increased in alcohol fed-mice compared with control mice, however, RSV stimulated AMPK activity and inhibited lipinl expression. This suggested AMPK and lipinl may be involved in the effects of RSV on AFL.3. RSV improved lipid accumulation in HepG2 cells induced by alcohol and oleic acidTo induce excessive lipid accumulation, HepG2 cells were exposed to control media supplemented with 100μM oleic acid and 87mM alcohol for 48 hours, with media being exchanged for fresh media after 24 hours. Oil red O staining and measurement of intracellular TG were used to evaluate the vitro model and observe the effects of RSV on the lipid accumulation in HepG2 cells induced by alcohol and oleic acid. Oil red O staining demonstrated oleic acid plus alcohol administration contributed to significant lipid accumulation in HepG2 cells, the intracellular lipid droplets were reduced significantly by RSV treatment. Measurement of intracellular TG showed that the level of TG significantly increased in HepG2 cells induced by alcohol and oleic acid, treatment with RSV led to a remarkable decrease in TG contents.These results suggested that RSV improved lipid accumulation in HepG2 cells induced by alcohol and oleic acid.4. Resveratrol ameliorates lipid accumulation in HepG2 cells induced by alcohol and oleic acid by activating AMPK-Lipinl signalingTo further determine whether RSV prevented HepG2 cells lipid accumulation via AMPK-Lipinl signaling pathway, we used the AMPK agonist (AICAR) and inhibitor (Compound C) to activate or suppress AMPK activity for detection of the expression of p-AMPK and lipin1. The results showed the level of p-AMPK significantly decreased lipinl expression increased significantly in HepG2 cells of model group. RSV significantly increased phosphorylation of AMPK levels and decreased the lipinl expression consistent with AICAR administration. However, Compound C administration suppressed the effects of RSV.These results suggested that resveratrol ameliorates lipid accumulation in HepG2 cells induced by alcohol and oleic acid by AMPK-Lipin1 signaling.