Effect And Mechanism Of Rosmarinic Acid Against H₂O₂-induced Oxidative Damage In LO2 Cells

Along with the changes in people’s lifestyle and dietary structure,the incidence of non-alcoholic fatty liver disease（NAFLD）is continuously increasing nowadays.NAFLD has been a global public health problem,which ranges from simple steatosis,nonalcoholic steatohepatitis,fibrosis,liver cirrhosis to end-stage hepatocellular carcinoma（HCC）,but there is no pharmacological agent for its treatment.Oxidative stress has been considered as a key factor in the pathophysiology of NAFLD.Therefore,the regulation based on oxidative stress could be an effective strategy to interfere with the pathological processes of NAFLD.Rosmarinic acid is a potent antioxidant from Rosmarinus officinalis.Reports have showed RA could ameliorate liver damage and protect liver in oxidative stress-induced oxidative stress in vivo and vitro.However,the cellular and molecular mechanisms under oxidative stress are not fully understood.The current study was conducted to investigate the cytoprotective effect of RA through the cell model of oxidative damage induced by H₂O₂,and further elucidated its new potential mechanism of signal transduction in LO2cells.Groups were divided in experiments as follows:（1）Control:Normal control group（2）Vit.C:Positive control vitamin C+H₂O₂ group（3）H₂O₂:H₂O₂ damage group（4）RA（RA0.25、RA1.00、RA5.00）:Rosmarinic acid（0.25,1.00,5.00μM）+H₂O₂ group.LO2 cells were pretreated with DMEM medium containing RA（0.25,1.00 and 5.00μM）or VC（100μM）for 24 h,and then stimulated with H₂O₂（400μM）for 6 h.Cell viabilities of LO2 cells were assayed by CCK-8 in each group.Then the ROS production,cell apoptosis,mitochondrial inner transmembrane potential and cell cycle arrest in LO2 cells were assayed by flow cytometry.And the ROS in cells were stained with DCFH-DA,cell apoptosis in cells was stained with DAPI andβ-galactosidase in cells was stained withβ-galactosidase staining liquids,then the above three different treatment cells were observed with a fluorescence microscope.Furthermore,we determined the redox-sensitive protein expression of MAPK pathway（JNK,ERK,p38）,Nrf2 pathway（Nrf2,NQO1）,and mitochondrial apoptotic pathway（Bcl-2,caspase-3,,Bid,Bax）.The results represented as follows:compared with the H₂O₂-induced damage group,the cell viabilities were increased in the RA treatment group.Besides,the percentage of cell apoptosis,the ROS level,mitochondrial inner transmembrane potential,the activity ofβ-galactosidase and the percentage of G₂/M cell cycle arrest were all decreased.Results also showed that the relevant protein expression of MAPK pathway,Nrf2 pathway,and mitochondrial apoptotic pathway were activated in the RA treatment group,which might provide the new therapeutic targets for NAFLD.In summary,RA could decrease H₂O₂-induced oxidative damage through decreasing G₂/M cell cycle arrest and activating of the MAPK,Nrf2,and mitochondrial apoptotic signaling pathways,which might perform the possible mechanisms for the protective effect of RA against H₂O₂-induced oxidative stress in LO2 cells.Based on previous experiments,these findings provided a new evidence to support RA as a potential candidate for the prevention or treatment of NAFLD.