695 18ß-Glycyrrhetinic Acid Prevents Free Fatty Acid-Induced Hepatic Lipotoxicity by Upregulating GRP78 Expression

Abstract

Free fatty acid (FFA)-induced hepatic lipotoxicity plays a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Inhibition of FFA-associated hepatic toxicity represents a potential therapeutic strategy. Recently, it was reported that FFA-induced hepatic injury is closely linked to the ER stress response and autophagy activities. Glycyrrhizin (GL), the major bioactive component of licorice root extract, is a traditional Chinese medicine and has been used to treat hepatitis to reduce liver inflammation and hepatic injury for many years. However, the underlying cellular mechanism responsible for its positive clinical effects has not been elucidated. 18β-glycyrrhetinic acid (GA) is the biologically activemetabolite of GL. We have previous reported that GA prevents FFA-induced hepatic lipotoxicity by stabilization of lysosomal membrane and inhibition of cathepsin B expression and enzyme activity in hepatocytes. Our preliminary studies indicate that palmitic acid (PA) dose-dependently activates the ER stress response in hepatocytes. The aim of the current study was to determine whether GA prevents PA-induced hepatic injury through modulating ER stress response and autophagy activities in hepatocytes. Methods: Rat primary hepatocytes were treated with 0.25 mM PA in the presence of GA (0-10 μM) for various time periods. The intracellular lipid accumulation was detected by nile red staining. The expression levels of ER stress marker genes (GRP78, CHOP, XBP-1, ATF-4) were determined by real-time RTPCR and Western blot analysis. Activation of autophagy was determined by measuring the conversion of microtubule-associated protein 1 light chain 3 (LC3) without or with 2 htreatment of lysosomal inhibitors using Western blot analysis. The mRNA expression levels of key genes involved in hepatic lipid metabolism were assessed using real-time RT-PCR. Lentiviral shRNA was used to knock down GRP78 expression. Results: In rat primary hepatocytes, GA increased GRP78 mRNA and protein levels by 53% and 28%, respectively, over control cells. In addition, GA induced autophagy activities and reduced intracellular lipid accumulation. Furthermore, GA significantly reduced the expression of SREBP-1/ SREBP-2-target genes, i.e. fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD-1), 3hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAR), and LDL receptor (LDL-R). GAmediated protective effect against PA-induced hepatic lipotoxicity was eliminated by downregulation of GRP78 expression using lentiviral shRNA. Conclusion: FFA-induced hepatic lipotoxicity is strongly related to the activation of hepatic ER stress. Regulation of GRP78 expression may represent an important mechanism by which GA prevents PA-induced hepatic lipotoxicity.