Salvianolic Acid B Protects Against Acute Ethanol-induced Liver Injury Through SIRT1-mediated Deacetylation Of P53in Rats

Background: Inflammation, oxidative stress, hepatocyte apoptosis, and necrosisare involved in the pathogenesis of ethanol-induced acute liver injury. It is now clearthat acute alcohol binges can be acutely toxic not only to the liver, but also contribute tothe chronicity of alcoholic liver disease (ALD). Sirtuin1(SIRT1) is a NAD+-dependentdeacetylase, studies have confirmed that SIRT1plays an important role in alcoholicliver disease. Salvianolic acid B (Sal B), one of the major water-soluble components ofthe Radix Salvia miltiorrhiza, has been widely used for treating many types of diseasesincluding hepatic, lung and renal diseases. It is reported that Sal B is effective ininhibiting apoptosis of various cell types. But the effect of salvianolic acid B in theregulation of SIRT1signaling pathway in acute ethanol-induced liver injury has notbeen reported.Objective: To investigate the effect of Salvianolic acid B on acute ethanol-inducedliver injury and explore the role of SIRT1in this process.Methods:1. Male Wistar rats underwent intragastric administration of Sal B (15or30mg/kg) once a day for3days. Ethanol, at dose of6g/kg, was also intragastricallyadministered to the rats every12hours for3times to induce acute liver injury. Theexperimental rats were randomly divided into five groups. The first group was thevehicle control and the rats in the second group were just given Sal B (30mg/kg). In thethird group, rats were treated with alcohol merely. In the fourth and fifth groups, Rats were pretreated with Sal B at a dose of15mg/kg and30mg/kg respectively. Ten hoursafter the last alcohol treatment, all animals were euthanized and the liver and bloodwere harvested for further analysis. The biochemical evaluations of serum transaminase,IL-6, TNF-α, GSH and GSH-PX were performed using commercially available kits andthe operation was carried out according to manufacturer’s instructions.sed Western blotanalysis was employed to determine the expressions of SIRT1, p53, acetyl-p53, PGC-1a,NF-κB, Bcl-xL and Cleaved caspase-3. The reverse transcription-PCR analysis wasemployed to determine the gene expression of SIRT1. Hepatocyte apoptosis wasdetected by TUNEL staining.2. In HepG2cells, the cell viability of the corresponding HepG2cells which arepretreated to different concentrations of Sal B (0.5μM,2μM,8μM) and then exposed toEtOH (200mM) were detected. The effect of Sal B on SIRT1expression and theexpression of p53, acetyl-p53and PGC-1a were examined while SIRT1was knockeddown by siRNA transfection. Western bolt analysis was employed to determine theexpression of SIRT1with the pretreatment of Sal B for different times (0h,1.5h,3h,6h)and dosages (0.5μM,2μM,8μM). Furthermore, we knocked down SIRT1expressionwith siRNA and then detected the effects of Sal B on the SIRT1and p53, acetyl-p53expressions by western blot.Results: In vivo, pretreatment with Sal B significantly reduced acuteethanol-induced elevation in the activities of serum ALT, AST and the increase of serumTNF-α, IL-6. Meanwhile, Sal B pretreatment ameliorated the increase of NF-κB andCleaved caspase-3expression caused by acute ethanol exposure, while Bcl-xLexpression was elevated. Moreover, pretreatment with Sal B significantly increased theexpression of SIRT1, which was closely associated with the down-regulation ofacetyl-p53at protein levels. In vitro, Sal B pretreatment increased SIRT1expression ina time and dose-dependent manner. However, knockdown of SIRT1using specificsiRNA blocked the effect of Sal B in up-regulation of SIRT1. Furthermore, blockade ofSIRT1significantly reversed the lower expression of acetyl-p53and Sal B-induceddown-regulation of acetyl-p53was also attenuated. Conclusion: Sal B may be a potent activator of SIRT1and can alleviate acuteethanol-induced liver injury through SIRT1-mediated repression of acetyl-p53pathway.