Toll-Like Receptor4(TLR4)-derived Reactive Oxygen Specics Contribute To Acute Alcohol-induced Hepatic Lipid Accumulation

Objective Alcohol is widely consumed around the world. Alcoholic fatty liver is a relatively benign state of ALD and excessive lipid accumulation in liver is the hallmark of alcoholic fatty liver. The elevation of fatty acid and triglyceride (TG) syntheses is main cause of excess lipid accumulation in the development of alcoholic fatty liver Sterol-regulatory element binding protein (SREBP)-1is the most important transcription factor that regulates the expression of genes for fatty acid and TG syntheses. Numerous reports support the important roles of hepatic SREBP-1in the processs of alcoholic fatty liver. A recent study showed that TLR4was involved in the process of chronic alcoholic fatty liver disease.To investigate the role of toll-like receptor4(TLR4)-derived reactive oxygen species or inflammatory cytokines contribute to acute alcohol-induced sterol-regulatory element binding protein (SREBP)-1activation and lipid accumulation.Methods The present study included four separate experiments. In experiment1, To investigate the role of a single-dose alcohol treatment in activation of sterol-regulatory element binding protein (SREBP)-1and accumulation of lipid, twelve of each male ICR (TLR4+/+), C3H/HeN (TLR4+/+) and C3H/HeJ (TLR4-/-) mice were randomly divided into alcohol treated group and control group. After fasted for two hours, the two groups were treated with4mg/kg alcohol and the same amount of saline via intragastric administration, respectively. All mice were sacrificed after12h from administration. Blood serum was collected for serum TG measurement. Liver was collected for measuring the ratio of liver weight to body weight. Some liver tissue was frozen-fixed for Oil red O staining to examine lipid accumulation and the rest was kept at-80℃. qPCR was used for analyzing the level of liver fatty acid, and downstream target genes fas, ace, scd-1, dgat-2mRNA of transcription factor SREBP-lc. TG kit was used for determining the content of TG in liver. In order to further study the dynamic effect of a single-dose of alcohol on the level of STEBP-lc,20of each male ICR (TLR4+/+)、 C3H/HeN (TLR4+/+) and C3H/HeJ (TLR4-/-) mice were randomly divided into five groups, control group and alcohol treated0.5h,2h,6h, and12h. All the mice were fasted. The alcohol treated groups were administrated intragstrically with4g/kg alcohol after fasted for13.5h,12h,8h, and2h, respectively. All the mice were sacrificed after fasted14h. Liver was collected for western blot test. In experiment2, to study the role of a single-dose alcohol exposure in activation of liver signal PI3K/Akt,12male TLR4+/+mice were randomly divided into two groups, alcohol treated group and control group. After fasted8h, alcohol treated group was given4g/kg alcohol for one-time through intragastric administration. Controlled group was given equal volume of saline. All mice were sacrificed six hours later after administration. Blood serum was collected for determining the level of insulin and glucose. Liver tissue was collected and kept under-80℃for analyzing the lever of liver irs-1and irs-2mRNA via qPCR. Twenty of each male TLR4+/+and TLR4-/-mice were divided into5groups randomly, control group and alcohol treated0.5h,2h,6h, and12h. Alcohol treated groups were administrated intragstrically with4g/kg alcohol after fasted for13.5h,12h,8h, and2h, respectively. Controlled group was given equal volume of saline. All the mice were sacrificed after fasted14h. Liver was collected for western blot test to study the effect of single-dose alcohol in the level of hepatic phosphorylase Akt and Akt total protein. In experiment3, in order to understand the effect of single-dose alcohol treatment on hepatic TLR4signal, twenty of each male ICR (TLR4+/+)、C3H/HeN (TLR4+/+) and C3H/HeJ (TLR4-/-) mice were randomly divided into5groups, control group and alcohol treated0.5h,2h,6h, and12h. All the mice were fasted. The alcohol treated groups were administrated intragstrically with4mg/kg alcohol after fasted for13.5h,12h,8h and2h, respectively. Controlled group was given equal volume of saline. All the mice were sacrificed after fasted14h. Collected liver homogenates for studying the level of TNF-α in liver by ELISA. The rest of liver tissue was prepared for determining the levels of hepatic MyD88total protein, phosphorylase IκBα and NF-κB p65nucleoprotein by Western blot. In experiment4, to further study TLR4induced reactive oxygen in a single-dose induced SREBP-1c activation and lipid accumulation, Twelve of each male TLR4+/+and TLR4-/-mice were divided into two groups randomly, control group and alcohol treated group. All the mice were fasted for12h, and then were administrated intragstrically with4g/kg alcohol and equal volume of saline for alcohol treated group and control group, respectively. The mice were sacrificed2h after administrated. Liver tissue was collected and kept under-80℃for analyzing the level of mRNA of NADPH oxidase subunit hepatic p67phox、gp91phox、p22phox and p47phox via qPCR. For the further study in the effect of PBN in alcohol induced lipid accumulation, sixteen male ICR mice were randomly divided into four groups, alcohol treated group, alcohol-PBN group, PBN group, and control group. Alcohol treated group was treated with4g/kg alcohol via intragastric administration. Control group was given the same volume of saline. Alcohol-PBN group was treated via intragastric administration with PBN100mg/kg30min before given alcohol, and was given PBN again4h after treated with alcohol. PBN group was treated with the PBN100mg/kg at the same time with Alcohol-PBN group but without given alcohol. The mice were sacrificed after given alcohol for12h. liver tissue was collected and some were frozen-fixed for Oil red O staining. The rest of liver tissue was kept under-80℃for analyzing the content of hepatic TG. The level of SREBP-1c nucleoprotein was determined with Western blot. Results Hepatic TLR4signaling was activated in alcohol-treated ICR (TLR4+/+) and C3H/HeN (TLR4+/+) but not C3H/HeJ (TLR4-/-) mice. Correspondingly, an obvious lipid accumulation was observed in ICR (TLR4+/+) and C3H/HeN (TLR4+/+) but not C3H/HeJ (TLR4-/-) mice. The level of hepatic mature SREBP-1was increased in alcohol-treated ICR (TLR4+/+) and C3H/HeN (TLR4+/+) but not C3H/HeJ (TLR4-/-) mice. Although a single dose of alcohol did not increase serum insulin level and hepatic insulin receptor substrate (irs)-1and irs-2expression, hepatic PI3K/Akt signaling was activated in alcohol-treated C3H/HeN (TLR4+/+) mice but not C3H/HeJ (TLR4-/-) mice. A single dose of alcohol did not increase the expression of hepatic inflammatory cytokines. Interestingly, the expression of hepatic p67phox and p9lphox, two NADPH oxidase subunits, was elevated in alcohol-treated C3H/HeN (TLR4+/+) but not C3H/HeJ (TLR4-/-) mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against acute alcohol-induced hepatic SREBP-1activation and lipid accumulation.Conclusions TLR4-derived reactive oxygen species rather than inflammatory cytokines contribute to acute alcohol-induced hepatic SREBP-1activation and lipid accumulation.