Preliminary Study On The Protective Effect And Mechanism Of Maslinic Acid On H₂O₂-induced Oxidative Injury In BRL-3A Cells

Objective: An oxidative injury model in Rat hepatic cell line BRL-3A was induced by hydrogen peroxide（H₂O₂）,the protective effect of maslinic acid（MA）was explored on this model,and the mechanism was studied preliminarily.Methods: 1.MA dose screening: BRL-3A cells were seeded in 96-well cell culture plates,cultured for12 h,and different concentrations of MA were added,the cell viability was detected by MTT.The control group,H₂O₂ group（model group）,H₂O₂+MA group were set and the cell viability was assayed by MTT.2.Antioxidative effect of MA on H₂O₂-stimulated BRL-3A cells was explored: The control group,the model group and H₂O₂+MA group were designed,the activities of LDH,SOD,CAT as well as the contents of GSH and MDA were detected by micro-enzyme-linked method.The intracellular ROS levels were measured using DCFH-DA.3.The mRNA and protein levels of Nrf2,HO-1 were determined: The control group and different concentrations of MA groups were set,the protein expression of Nrf2 and HO-1 were detected by Western Blot.The control group and different times of MA groups were designed,the protein expression of Nrf2 and HO-1 were detected by Western Blot.The control group,the model group,H₂O₂+MA group and MA alone group were set,the mRNA and protein expression of Nrf2 and HO-1 were detected by RT-PCR and Western Blot.4.The relative antioxidative mechanism of MA was investigated:The control group,the model group,H₂O₂+MA group and MA alone group were set,the protein phosphorylation levels of p38,JNK and ERK were detected by Western Blot.The control group,the model group,H₂O₂+MA group,H₂O₂+MA+SB203580 group and H₂O₂+MA+SP600125 group were set,the cell viability was determined by MTT.Results: 1.Compared with the control group,there was no significant change in cell viability when BRL-3A cells were stimulated by different concentrations of MA.The result showed that MA at dose of0³0 μmol/L had no obvious toxicity to BRL-3A cells.Compared with the model group,the cell viability could be increased significantly by MA when the H₂O₂-induced BRL-3A cells were preincubated with different concentrations of MA.2.Compared with the model group,the activities of SOD,CAT and the content of GSH could be increased significantly,the activity of LDH,the content of MDA and the level of ROS could be decreased significantly.3.With the increase of time and concentration,the protein expression of nucleus Nrf2 and HO-1 could be increased significantly,the protein expression of cytoplasm Nrf2 could be decreased significantly.Compared with the model group,the mRNA expression of Nrf2 and HO-1 could be increased significantly.The protein expression of nucleus Nrf2 and HO-1 could be increased significantly,the protein expression of cytoplasm Nrf2 could be decreased significantly.4.Compared with the model group,the protein phosphorylation levels of p38 and JNK could be decreased significantly.Compared with the MA pretreatment group,the cell viability could be decreased significantly when SB203580 and SP600125 treated.Conclusion: 1.In H₂O₂-induced BRL-3A cells,the activities of SOD and CAT,the GSH content were increased,but the activity of LDH,the levels of ROS and MDA were reduced by MA.2.The nuclear translocation of Nrf2 could be promoted.The expression of HO-1 both in mRNA level and protein level was up-regulated.3.The Nrf2 and p38,JNK MAPK siganal pathway could be activated by MA.Therefore,H₂O₂-induced oxidative injury in BRL-3A cells could be protected by MA,and its mechanism was probably associated with activating p38 and JNK MAPK siganal pathway as well as Nrf2/HO-1 siganal pathway.